Wiskott-Aldrich Syndrome (WAS) is a rare X-linked primary immunodeficiency affecting approximately 1 in 100,000 live XY births in North America and is caused by a mutation to the WAS gene which is expressed across hematopoietic lineages. The WAS protein (WASp) plays a role in regulating actin polymerization. On a cellular level, there are a variety of effects of a lack of WASp or expression of a dysfunctional WASp protein for patients including issues with migration, adhesion, chemotactic response, phagocytosis, activation, and proliferation across different cell types in addition to reduced platelet size and output. This can lead to several systematic effects for the patients however because mutations to the WAS gene are not limited to one location or type there is a great amount of variability between patient symptoms making it challenging to diagnose. Major symptoms include frequent and recurrent infections, uncontrolled bleeding episodes, issues associated with autoimmunity, and malignancy, the most common form being lymphoma. Without treatment, the life expectancy of an individual diagnosed with WAS is 14 years of age, and the only curative treatment strategy available is hematopoietic stem and progenitor cell transfer (HSPCT). If not performed with an HLA-identical donor, which is available to less than 10% of patients, and within the first two years of life, the risk of graft versus host disease (GvHD) increases drastically for the patient. A gene therapy using autologous and genetically corrected CD34+ cells would be advantageous to the patients due to a reduction in preparative conditioning, immunosuppressive aftercare, and the risk of GvHD. CSL Behring is currently in the development of a lentiviral gene therapy to fulfill this gap in care, however, to develop the assays required to assess and characterize the drug substance usually an uncorrected patient sample is compared with a gene-edited sample. The limitation here is that due to the risk of infection and bleeding patient sample is very limited and therefore the development of a mock patient sample is necessary for early development. The goal of the project is to develop a WAS-KO protocol utilizing CRISPR/Cas9 and its characterization.

In the growing landscape of innovative non-viral delivery vehicles, polymeric and lipid nanoparticles remain at the forefront for their versatility in encapsulating a variety of therapeutic payloads. This thesis investigates their potential for facilitating the transport of nucleic acid components into cells, with a focus on targeted delivery to the liver and brain. To achieve this, we address key considerations including the composition of the delivery vector, the nature of the therapeutic cargo, and the chosen delivery route. The challenge of targeted delivery to specific organs or cell types, i.e. hepatocytes or neurons, is addressed through rational design and development of libraries of nanoparticulate systems tailored for nucleic acid therapeutics. Although liver gene editing using non-viral systems has been extensively studied, oral delivery for liver targeting remains challenging due to the mucosal barrier. To that end, we explore intraduodenal delivery as a strategy to bypass the mucosal barrier and target the liver. Furthermore, insights from collaborative research with the Mao lab at Johns Hopkins University reveal that tuning the composition of lipid nanoparticles (LNPs) can influence their preferential targeting of specific cell types. Leveraging this, we employed an in vitro library screening and machine learning approach to identify populations of LNPs capable of preferentially transfecting hepatocytes. The efficacy of these LNPs in liver gene editing is then evaluated through “cluster-mode” screening in vivo, and therapeutic efficacy was demonstrated using a proof-of-concept in vivo model for PCSK9 and ANGPTL3 knockdown, resulting in 27% serum cholesterol knockdown. In addition to liver-targeted gene delivery, this thesis also investigates the potential of polymeric and lipid nanoparticles for delivering nucleic acid therapeutics to the brain. However, overcoming the blood-brain barrier (BBB) is crucial for systemic delivery to the brain. To circumvent the BBB, we explored two methods: intracranial injection and theranostic ultrasound (THUS)-mediated temporary opening of the BBB. While intracranial injection achieves localized gene editing, THUS offers a non-invasive approach for transient and widespread BBB opening. Utilizing the previously validated in vitro screening and machine learning approaches for chitosan-grafted bPEI (CS-PEI) and lipid nanoparticle (LNP) carriers with tunable compositions, we assessed their efficacy in systemic gene delivery to the brain, and specifically their capability in preferentially transfecting neuronal cells over hepatocytes. Subsequently, we validated their efficiency via intracranial administration using the Ai14 reporter mouse model and observed up to 20% gene editing of the targeted cross-sectional area of the brain hemisphere using the top-performing cluster. Through comprehensive investigations into both brain and liver gene delivery, this thesis aims to contribute to the advancement of non-viral nanoparticle-based gene therapy strategies for treating a range of cholestatic liver diseases and hereditary neurodegenerative diseases.

In vitro systems like those used for in vitro embryo production are invaluable for our understanding of embryogenesis and the processes that regulate it. However, extensive research has also highlighted that in vitro produced embryos negatively differ from their in vivo counterparts in various ways. Not surprisingly, there is ~20% decrease in pregnancy success from pregnancies established using in vitro produced embryos. Therefore, much research has relied on attempting to produce a better in vitro embryo that more closely resembles their in vivo counterparts. Our laboratory has investigated this by supplementing a cytokine, interleukin-6 (IL6), during in vitro embryo culture. My dissertation work expands upon those initial efforts by answering more detailed questions related to the biological role of IL6 during cattle embryogenesis. In the work presented herein, IL6 supplementation during in vitro culture was able to transform the transcriptome of resulting conceptuses post embryo transfer. The transcriptome of these conceptuses included an abundance of genes associated with survival. Indeed, we witnessed IL6-treated conceptuses resulted in a 20% increased survival rate and were longer than their non-treated counterparts. In the second research project, we employed CRISPR-Cas9 genome editing technology to understand the embryo phenotype after part of the IL6 receptor responsible for signal transduction, interleukin-6 signal transducer (IL6ST), is disrupted. We discovered that IL6ST is required for development before the blastocyst stage. In addition, IL6ST disrupted blastocysts, presumed to contain wildtype, presented with severe, abnormal morphology. Not only did this group of embryos have decreased ICM and TE cell numbers, but they also had an increased occurrence of cells within the TE region that were negative for its traditional marker, CDX2. This suggests IL6ST is likely involved in a pathway responsible for determining cell fate identity at the blastocyst stage. Collectively, IL6 in cooperation with IL6ST, is a key controller of embryogenesis in cattle. / Doctor of Philosophy / There are major events that an embryo must successfully advance from to continue development to form into an organism capable of survival after birth. Over 30% of pregnancies in cattle and humans will fail within the first 30 days of gestation. This time period coincides with several key developmental events that ultimately modify the morphology of the growing embryo. Our laboratory primarily focuses on embryo development around the blastocyst stage. If an embryo advances to this stage, it has a greater likelihood of maintaining viability. Therefore, my dissertation research has focused on early embryonic development from the time of first cleavage (~day 2 of gestation) through embryo elongation (~day 15 of gestation), which encompasses the blastocyst stage. Within this time frame, I have been investigating embryonic effects after supplementation of a protein, interleukin-6 (IL6). Previously, our laboratory has identified IL6 to cause favorable impacts on the developing embryo, but its mode of action was unknown. Therefore, my dissertation research has investigated the mechanistic actions of IL6, and its beta receptor subunit, interleukin-6 signal transducer (IL6ST). In my first research project, we discovered that supplementing IL6 during in vitro embryo culture resulted in increased embryo elongation and survival. In my second research project, we found IL6ST is an absolute requirement for embryo survival to the blastocyst stage. Together, these results indicate IL6 is a very important protein needed for sustained pregnancy viability.

A perda auditiva hereditária é uma característica com grande heterogeneidade genética. Mais de uma centena de genes já foram relacionados com a audição e, com o advento do sequenciamento massivo em paralelo, novas variantes têm sido identificadas como candidatas a causar surdez hereditária. Porém, estudos funcionais para verificação do efeito das mutações candidatas são necessários. Modelos animais permitem estudos funcionais eficientes para confirmação do efeito de mutações em genes candidatos, sendo uma ferramenta poderosa para compreender melhor os efeitos genéticos, moleculares, fisiológicos e comportamentais destas alterações. Previamente, foi identificada em nosso laboratório uma mutação de sentido trocado em um gene que codifica um coativador nuclear como principal candidata a causar surdez hereditária em uma família de São Paulo. A função principal deste gene é regular positivamente a transcrição gênica mediada por receptores nucleares. Contudo, não há dados na literatura sobre a expressão e o papel deste gene no sistema auditivo. Desta forma, tivemos como objetivo principal investigar a expressão do gene candidato em sistemas mecanossensoriais de camundongo e zebrafish. Detectamos, por meio de RT-PCR, a expressão do RNAm na cóclea inteira e no órgão de Corti associado à estria vascular de camundongos com idades P4, P10 e P16. Posteriormente, experimentos de qRT-PCR mostraram maior expressão nos estágios P10 e P16, em relação ao estágio P4, com parcela significativa da expressão gênica concentrada no órgão de Corti associado à estria vascular. Com relação à proteína, foi detectada, por meio de ensaios de imunofluorescência, a sua expressão nos cortes histológicos de cóclea de camundongos P4, P10 e P14, em várias estruturas diferentes da cóclea: membrana basilar, membrana de Reissner, órgão de Corti, estria vascular, limbo espiral e gânglio espiral. Experimentos de hibridação in situ em zebrafish inteiro foram realizados e a expressão do RNAm foi observada na orelha interna de larvas com idade 3 e 5 dias pós-fertilização (dpf) e de juvenis com 5 e 7 semanas pós-fertilização (spf). Nossos experimentos forneceram dados inéditos que sugerem um papel conservado deste gene no desenvolvimento do sistema auditivo de camundongos e no desenvolvimento e fisiologia do sistema auditivo de zebrafish. Para investigarmos se a falta de expressão do gene afeta o sistema auditivo realizamos experimentos visando à edição gênica do gene candidato por meio do sistema CRISPR/Cas9 em zebrafish. Dada a dificuldade para a padronização da técnica, não obtivemos resultados conclusivos durante o período de estudo. Nosso trabalho mostrou pela primeira vez a expressão do RNAm e da proteína no sistema auditivo de modelos animais, o que é fundamental para reforçar o potencial papel da mutação na surdez hereditária identificada na família. Desta maneira, contribuímos para a delineação de futuros experimentos funcionais que elucidem o papel deste gene e da mutação correspondente no desenvolvimento e na fisiologia do sistema auditivo / Hereditary hearing loss is a characteristic with high genetic heterogeneity. More than one hundred genes have been related to hearing and, with the advent of massive parallel sequencing, new variants have been identified as candidates for causing hereditary deafness. However, functional studies to verify the effect of candidate mutations are necessary. Animal models provide efficient functional studies to confirm the effect of mutations and candidate genes, being a powerful tool to understanding the genetic, molecular, physiological and behavioural effects of these genetic variants. Previously, a missense mutation in a gene coding a nuclear receptor coactivator has been identified in our laboratory as the main candidate for causing hereditary hearing loss in a family from São Paulo. The main function of this gene is to positively regulate gene transcription mediated by nuclear receptors. However, there is no data in the literature about its expression or about its function in the hearing system. Thus, we aimed to investigate its expression in mechanosensory systems of mice and zebrafish. Through RT-CPR, we have detected expression of the mRNA in whole cochlea and in organ of Corti associated to stria vascularis of P4, P10 and P16 mice. In addition, qRT-PCR revealed higher expression in P10 and P16 stages, compared to P4 stage, and that a significant fraction of the expression is concentrated in the organ of Corti associated to stria vascularis. Regarding the protein, immunofluorescence assays revealed expression of the coactivator in histological sections of P4, P10 and P14 mice cochlea, with fluorescencent signals in several structures: basilar mebrane, Reissner\'s membrane, organ of Corti, stria vascularis, spiral limbus and spiral ganglion. Whole-mount in situ hybridization assays were conducted in zebrafish, revealing the mRNA expression in the inner ear of 3 and 5 days-post-fertilization (dpf) larvae and of 5 and 7 weeks-post-fertilization (wpf) juveniles. Our experiments provided unprecedented data suggesting a conserved role of this gene in the development of the auditory system of mice and in the development and physiology of the auditory system of zebrafish. In order to investigate if the lack of the gene expression affects the auditory system, we performed experiments aiming genetic edition through CRISPR/Cas9 system in zebrafish. Given the difficulty to standardize the technique, we could not obtain conclusive results during the study period. Our work has shown for the first time the expression of the candidate gene mRNA and protein in the auditory in animal models, which is fundamental to reinforce the potential role of the mutation in the hearing loss identified in the family. Thus, we have contributed to the delineation of future functional experiments that will unveil the role of the gene and its corresponding mutation in the development and physiology of the auditory system

The largest U.S. cancer health disparity exists in prostate cancer, with African American men having the highest incidence and mortality rates. The present study evaluated the effects of ZIC2 and the underlying mechanisms in the E006 parental African-American cell line that produces tumors at accelerated growth rates because of the increase of ZIC2 genes in African-American males. We analyzed the experimental research that the overexpression of ZIC2 contributes to progression of prostate cancer. E006AA cells with overexpressed or suppressed ZIC2 were analyzed to determine phenotypic differences, PCR, cell proliferation and immunoblot assays. The expression levels of ZIC2 were analyzed by CRISPR-Cas9, Western blot and proliferation growth curves. We discovered using these experimental techniques to knockout ZIC2, reduced cell proliferation occurred. This research investigated the role of ZIC2 in prostate cancer progression and the effects of the loss or gain of function of ZIC2 by using CRISPR-Cas 9 genome editing technology.

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第22700号 / 医科博第115号 / 新制||医科||8(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 遊佐 宏介, 教授 近藤 玄, 教授 齊藤 博英 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The Cre/loxP system belongs to the family of site-specific recombinases (SSR) that can precisely modify DNA that is flanked by two target sites. The reaction outcome is dependent on the structure and orientation of the target sites and includes excision, inversion and exchange of a DNA fragment. The system is established for more than 30 years and is active in vitro and in vivo in several organisms. These characteristics make the Cre/loxP system the ideal tool for genome editing. However, the strict target site preference for loxP limits its use to basic research where the loxP target sites can be introduced beforehand at the anticipated genomic locus. Directed evolution strategies have overcome this limitation and allow to generate Cre-like recombinases with altered DNA specificity. During this work, I developed the first dual-recombinase system based on evolved recombinases. Using two instead of one recombinase expands the targetability of the human genome by being more flexible in the target site search. After the identification of suitable target sites, I could show an evolved dual-recombinase system that can be used for excision and inversion of a human genomic locus. The recombinase mediated inversion reaction corrected a large genomic inversion that is frequently found in patients with severe Hemophilia A. Only two days after treating human cells with the developed dual-recombinase system, RecF8, 30% inversion could be detected in a human cell line. Applying RecF8 in patient specific endothelial cells corrected around 9% of the inversion back to the wild type sequence, which would be sufficient to drastically improve the quality of life of affected individuals. This genomic correction lead to the expression of the F8 gene, which is inactive elsewise. It remains to proof that the transcriptional reactivation of the F8 gene allows for the production of the Factor VIII protein. Before using RecF8 in a clinical setting, an in vivo study in a suitable mouse model is necessary. This study introduces a dual-recombinase system and thereby broadens the use of designer recombinases for genome editing. Moreover, in a proof on concept experiment this study shows that recombinases can be applied to correct large disease-causing genomic inversions in human cells. Altogether, the use of recombinases for scarless genome editing comes a step closer to reality.

IntroductionPreimplantation Genetic Diagnosis (PGD) was developed in the 1990s and has been used since to diagnose and discard embryos with genetic conditions or chromosomal abnormalities. CRISPR-Cas9 was discovered in 2012 and has been used in research, but has not become clinical practice on humans yet. CRISPR-Cas9 could potentially be applied to treat and prevent genetic disorders.AimThe aim was to investigate the ethical dilemmas of each method through a set of research questions. The ethics of applying PGD according to Swedish guidelines and applying CRISPR-Cas9 on humans was investigated.MethodologyThis was not a systematic literature review. Instead, articles have been selected based on their explanation of each method and uniqueness or volume of ethical arguments surrounding each method, that is of relevance for the discussed issues.ResultsArguments in favour of PGD addressed among other things the somatic and psychological health of future children and parents along with the economical benefits. Arguments against PGD addressed different dilemmas of discarding an embryo and thereby a future individual. Arguments against CRISPR-Cas9 addressed technical limitations, our limited knowledge of genetics and more. Arguments in favour addressed benefits in clinical medicine and research.ConclusionsPGD according to Swedish guidelines was found to be ethically acceptable, since its restrictive use that have not given room for ethically dubious applications. CRISPR-Cas9 was found not to be safe enough for human applications at this moment due to technical limitations. If these were to be solved, caution and restraint must be urged.

京都大学 / 新制・課程博士 / 博士(医科学) / 甲第24885号 / 医科博第152号 / 新制||医科||10(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 河本 宏, 教授 濵﨑 洋子, 教授 上野 英樹 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

T cells modified for expression of Chimeric Antigen Receptors (CARs) were the first genemodified cell products approved for use in cancer immunotherapy. CAR-T cells engineered with gammaretroviral or lentiviral vectors (RVs/LVs) targeting B-cell lymphomas and leukemias have shown excellent clinical efficacy and no malignant transformation due to insertional mutagenesis to date. Large-scale production of RVs/ LVs under good-manufacturing practices for CAR-T cell manufacturing has soared in recent years. However, manufacturing of RVs/LVs remains complex and costly, representing a logistical bottleneck for CAR-T cell production. Emerging gene-editing technologies are fostering a new paradigm in synthetic biology for the engineering and production of CAR-T cells. Firstly, the generation of the modular reagents utilized for gene editing with the CRISPR-Cas systems can be scaled-up with high precision under good manufacturing practices, are interchangeable and can be more sustainable in the long-run through the lower material costs. Secondly, gene editing exploits the precise insertion of CARs into defined genomic loci and allows combinatorial gene knock-ins and knock-outs with exciting and dynamic perspectives for T cell engineering to improve their therapeutic efficacy. Thirdly, allogeneic edited CAR-effector cells could eventually become available as “off-the-shelf” products. This review addresses important points to consider regarding the status quo, pending needs and perspectives for the forthright evolution from the viral towards gene editing developments for CAR-T cells.